TAS2R supports odontoblastic differentiation of human dental pulp stem cells in the inflammatory microenvironment

Background Inflammatory microenvironment promotes odontoblastic differentiation in human dental pulp stem cells (hDPSCs), but the regulatory mechanisms remain unclear. In this study, we aimed to explore the role of TAS2R in odontoblastic differentiation of hDPSCs in the inflammatory microenvironment. Methods Microarray analysis was performed to explore the differential mRNA profiles in inflammatory and healthy pulp tissues from the patients. hDPSCs isolated from the healthy pulp tissues were stimulated by LPS, TNFα and IL-6, respectively, to verify the effect of TAS2R. The expression markers related to odontoblastic differentiation of hDPSCs were observed by qPCR and chemical staining methods. TAS2R10 was overexpressed or silenced to observe the effect on odontoblastic differentiation of hDPSCs under LPS stimulation. The G protein and intracellular Ca2+ were detected, respectively, by qPCR and Fluo-4AM Ca2+ fluorescent probe. Results The expression of TAS2R was significantly upregulated in the inflammatory pulp tissues. In vitro, 5 subtypes of TAS2R mRNA expressions including TAS2R10, TAS2R14, TAS2R19, TAS2R30 and TAS2R31 in hDPSCs increased under the stimulation of LPS, TNFα or IL-6. In odontoblastic differentiation medium, we found LPS, TNFα or IL-6 stimulation promoted odontoblastic differentiation of hDPSCs. TAS2R10 overexpression in hDPSCs significantly increased the expression markers related to odontoblastic differentiation, whereas TAS2R10 silencing revealed the opposite effect. Furthermore, G protein was activated, and at the same time, intracellular Ca2+ enhanced when TAS2R10 was overexpressed, but decreased when TAS2R10 was silenced. Conclusions This study demonstrated that TAS2R was found to be expressed in hDPSCs, and TAS2R promoted odontoblastic differentiation of hDPSCs by mediating the increase in intracellular Ca2+ via the G protein-coupled receptors (GPCR) conventional signaling pathway in inflammatory microenvironment, which may be a potential target for the development of effective conservative treatments for dental pulp repair.

cells (hDPSCs) to the site of injury and eventual differentiation into restorative dentin [1,4]. As the main cells of dental pulp repair, hDPSCs have the properties of mesenchymal stem cells with strong proliferative capacity, immunomodulatory properties and multidirectional differentiation potential [5]. hDPSCs even show remarkable neurogenic potential, since they originate from the neural crest [6,7]. Many studies have found that inflammatory microenvironment can promote the odontoblastic differentiation of hDPSCs in vitro [8][9][10]. However, the mechanism of this process remains unclear. Recent studies have shown that bitter-taste receptors type 2 (TAS2R) which belongs to the G protein-coupled receptors (GPCR) family, plays an important role in the differentiation of stem cells. As Seo et al. [11] reported, TAS2R induces neuronal cells differentiation of cancer stem cells, thereby suppressing cancer stemness in human neuroblastoma cells. Wölfle et al. [12] reported that TAS2R promotes keratinocytes differentiation. Interestingly, in gingival fibroblasts of periodontitis [13] and gut epithelial cell of intestinal inflammation [14], the TAS2R expression is increased. Chemicals secreted by Gram-negative bacteria may be direct ligand of TAS2R, such as acyl-homoserine lactones (AHLs) [15]. Under LPS (a virulence factor released by Gram-negative bacteria) stimulation in vitro, the expression level of TAS2R is enhanced in lung macrophages [16]. Additionally, in the preliminary experiment, we observed that TAS2R was expressed in dental pulp, and the expression of TAS2R increased in inflammatory pulp tissues. We hypothesized that TAS2R might be expressed in hDPSCs and played a role in the differentiation of hDPSCs under inflammatory microenvironment.
In this study, we investigated the differential expression profiles between inflammatory and healthy pulp tissues by microarray and validated the results using qPCR. Then, the effect of TAS2R on promoting odontoblastic differentiation of hDPSCs under the inflammatory microenvironment in vitro was detected. This study might reveal the mechanism of pulpitis repair and might provide a new idea for conservative treatment of pulpitis.

Collection of pulp samples
The inflammatory samples diagnosed as irreversible pulpitis according to the endodontic diagnosis system of the American Association of Endodontist [9] were obtained from the molar with deep caries and clinical symptoms were spontaneous pain, hot and cold irritation pain. The healthy samples were taken from non-carious impacted teeth. All samples used for study were taken from adults aged 18-28. Teeth with periodontitis were excluded. Samples were immediately frozen at −80 ℃ for subsequent experiments. Patients signed the informed consent. Approval was obtained from the Ethics Committee of Nanjing Stomatological Hospital, Medical school of Nanjing University.

Microarray analysis
Total RNA was extracted from the pulp tissues by RNeasy Kit (QIAGEN, USA) according to the manufacturer's instructions. Microarray analysis (OE Bio-tech, Shanghai, China) using inflammatory and healthy pulp tissues was performed. Differentially expressed RNAs were identified based on fold change ≥ 2.0 and P value ≤ 0.05. Afterwards, Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to determine the roles of these differentially expressed mRNAs.
Isolation, culture, flow cytometry identification and preconditioning of hDPSCs hDPSCs were isolated from the third molars of healthy adults aged 18-28 years by enzymatic digestion method [17]. Briefly, the pulp tissues were extracted and cut into tiny pieces and digested with 5 mg/mL collagenase type P (Roche, Switzerland) for 30 min at 37 ℃ to obtain a single-cell suspension. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, USA) at 37 °C and 5% CO 2 .
The third passage hDPSCs were identified via their surface antigen markers by flow cytometry. hDPSCs were resuspended and incubated in PBS (Gibco, USA) supplemented with 3% FBS for 45 min with primary antibodies against CD34, CD45, CD44 and CD146 [18]. Flow cytometer (BD FACSVerse, USA) was used for Flow cytometry analysis.

ALP and Alizarin red staining
After 7 days or 14 days of odontoblastic differentiation, alkaline phosphatase (ALP) activity was evaluated by ALP staining [22]. The number of mineralized nodules was detected by Alizarin red staining [23]. hDPSCs were fixed with 4% paraformaldehyde for 30 min and washed with deionized water for 3 times at room temperature. hDPSCs were then stained with ALP (Beyotime, Shanghai, China) and Alizarin red (BestBio, Shanghai, China) using the corresponding assay kit according to the manufacturer's instructions.

Statistical analysis
GraphPad Prism 7.0 software was used for One-way analysis of variance (ANOVA) and Student's t-test. All experiments were repeated at least three times. The experimental data were expressed as mean ± standard deviation, and P < 0.05 was considered statistically significant.

TAS2R was significantly upregulated in the inflammatory pulp tissues in mRNA profiles
Microarray technology could reveal the differential expression profiles between inflammatory and healthy pulp tissues, which might help us discover the genes or pathways that played a key role in inflammation promoting differentiation of hDPSCs. Thus, we collected a total of 4 pulp samples, including 2 inflammatory samples and 2 healthy samples for microarray analysis. Two inflammatory samples were obtained from the mandibular first molar. The average age was 25, including 1 male and 1 female. Two healthy samples were taken from the mandibular third molar. The average age was 22, including 1 male and 1 female. By extracting total RNA and measuring the RNA concentration, the purity, the quality of the 4 samples was tested and could be used for the subsequent experiments.
According to the analysis of differential mRNA profiles in inflammatory and healthy pulp tissues, the volcano plots showed 1727 differential genes between the two groups, of which 1128 were upregulated and 599 were downregulated (Fig. 1a). By GO enrichment analysis, the differentially up-or downregulated gene pathways were enriched including biological process,   (Fig. 1b). We also found that upregulated gene pathways were mainly involved in regulating cell proliferation, growth, metabolism, adhesion and other functions. And the top 1 upregulated gene pathway was associated with TAS2R (Fig. 1c). The KEGG analysis was used to explore which pathways might be associated with the differential genes, and the scatter plots showed that the pathways with the most abundant genes and the most significant differences were related to TAS2R transduction (Fig. 1d). Genes enriched in TAS2R pathway are shown in Table 1.

TAS2R was increased in hDPSCs under LPS, TNFα or IL-6 stimulation
As hDPSCs are the main cells of dental pulp repair under injurious stimuli, we investigated the expression of TAS2R in hDPSCs. hDPSCs were cultured in vitro and the expression of TAS2R in hDPSCs was detected by qPCR. The results showed that hDPSCs were successfully isolated and cultured from the pulp of impacted teeth, and hDPSCs were positive for stem cell surface markers CD44, CD146 and negative for CD34, CD45 by flow cytometry analysis (Fig. 2a-d). qPCR results showed that there were mRNA expressions of TAS2R10, TAS2R14, TAS2R19, TAS2R30 and TAS2R31 in hDPSCs (Fig. 2e).
To verify the microarray results, we stimulated hDP-SCs with LPS, TNFα and IL-6 to mimic an inflammatory microenvironment, and detected the expression of TAS2R in hDPSCs by qPCR. The dominant bacteria in pulpitis are Gram-negative anaerobic bacteria [24]. LPS, a virulence factor from Gram-negative bacteria, is released at high levels in pulpitis [25]. In addition, TNFα and IL-6 are also highly expressed in the pulpitis [26]. Therefore, LPS, TNFα and IL-6 were selected in our study to stimulate hDPSCs to produce an inflammatory microenvironment. The results showed that the mRNA expression of TAS2R10, TAS2R14, TAS2R19, TAS2R30 and TAS2R31 increased after stimulation of hDPSCs with 1 µg/mL LPS, 10 ng/mL TNFα and 10 ng/mL IL-6 for 12 h and 24 h, respectively (Fig. 2f-j). Since TAS2R10 expression was highest in hDPSCs and increased significantly under the inflammatory microenvironment. TAS2R10 was selected for the subsequent experiments.

TAS2R was involved the odontoblastic differentiation of hDPSCs in the inflammatory microenvironment
To demonstrate that the inflammatory microenvironment promoted differentiation of hDPSCs, hDPSCs were stimulated with 1 µg/mL LPS, 10 ng/mL TNFα or 10 ng/ mL IL-6 in ODI for 7 days and 14 days, respectively. The results showed that LPS, TNFα and IL-6 could improve ALP activity (Fig. 3a-j) and enhance the expression markers related to odontoblastic differentiation including ALP, RUNX2, DMP-1 and DSPP (Fig. 3k-n). And 1 µg/ mL LPS was selected to produce inflammatory microenvironment for subsequent experiments.
Meanwhile, the differential expressions of ALP, RUNX2, DSPP and DMP-1 were detected by microarray between inflammatory and healthy pulp tissues. The results showed that the mRNA expressions of ALP and RUNX2 were upregulated, but DMP-1 expression was downregulated in inflammatory pulp tissues. There was no difference in DSPP expression (P > 0.05) (Fig. 3o).
In the above experiments, we found that inflammatory microenvironment (such as LPS-induced inflammatory microenvironment) could promote the odontoblastic differentiation of hDPSCs. And the TAS2R expression (such as TAS2R10) was increased under LPS stimulation. To examine whether the LPS-induced differentiation of hDPSCs was dependent on TAS2R10, hDPSCs were co-treated with LPS and the TAS2R10 agonist QUN (50 µM) or siRNA-TAS2R10 transfection. Results showed that TAS2R10 overexpression in hDPSCs significantly enhanced the ALP activity and mineralized nodules formation, whereas TAS2R10 silencing revealed the opposite effect (Fig. 4a-j). The results of qPCR demonstrated that the expressions of ALP, RUNX2, DMP-1 and DSPP were remarkably increased when TAS2R10 was overexpressed, but decreased when TAS2R10 was silenced (Fig. 4k-n). These data revealed that TAS2R10 played a positive regulatory role in odontoblastic differentiation of hDPSCs.

TAS2R positively regulated intracellular Ca 2+ and promoted the odontoblastic differentiation of hDPSCs under LPS-stimulation
Gα and Gβ are two subunits of G protein, which are downstream molecules of TAS2R.
The mRNA expressions of Gα and Gβ were detected by qPCR. The results showed that the expression levels  (Fig. 4o, p). An increase in intracellular Ca 2+ is a typical downstream effect mediated by TAS2R. Previous studies have reported that intracellular Ca 2+ positively regulates the odontoblastic differentiation of hDPSCs [27,28]. Therefore, Fluo-4 AM Ca 2+ fluorescent probe was used to evaluate the intracellular Ca 2+ in this study. The results showed that the intracellular Ca 2+ was increased in the ODI group compared with the Control group. After LPS stimulation, the intracellular Ca 2+ further enhanced. The intracellular Ca 2+ were the highest in ODI + LPS + QUN group, but decreased in ODI + LPS + siRNA group (Fig. 4q). It was suggested that activation of TAS2R10 lead to an increase in intracellular Ca 2+ .

Discussion
Dental caries is the most important factor that cause inflammation of dental pulp and may eventually lead to the pulp necrosis [29]. However, the emerging evidences indicate that inflammation is an essential stage in pulp tissue repair and regeneration [30]. When the dental pulp is inflamed, hDPSCs migrate to the site of injury and differentiate into odontoblasts, forming reparative dentin and protecting pulp vitality [31]. Therefore, the more thoroughly study on the mechanisms of odontoblastic differentiation of hDPSCs, the more reliable the conservative treatment of pulpitis will be. Huang et al. [9] found that inflammationinduced overexpression of microRNA-223-3p regulates Fig. 2 Flow cytometry results of the surface marker and the expression of TAS2R in hDPSCs stimulated by LPS, TNFα or IL-6. a-d hDPSCs were positive for CD44, CD146 and negative for CD34, CD45. e TAS2R10, TAS2R14, TAS2R19, TAS2R30 and TAS2R31 were found to be expressed in hDPSCs. f-j The mRNA expressions of TAS2R10, TAS2R14, TAS2R19, TAS2R30 and TAS2R31 in hDPSCs after 12 h and 24 h stimulation with 1 µg/mL LPS, 10 ng/ mL TNFα and 10 ng/mL IL-6, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 Fig. 3 LPS, TNFα or IL-6 promoted odontoblastic differentiation of hDPSCs. a-j hDPSCs were cultured in ODI containing with 1 µg/ml LPS, 10 ng/ mL TNFα and 10 ng/mL IL-6, respectively, for 7 days and 14 days and then stained with ALP. hDPSCs in DMEM medium were used as control. k-n The mRNA expressions of ALP, RUNX2, DMP-1, DSPP were analyzed by qPCR. o The mRNA expression fold changes of ALP, RUNX2, DMP-1 in microarray. *P < 0.05; **P < 0.01; ***P < 0.001 odontoblastic differentiation of hDPSCs by targeting SMAD3. Xu et al. [19] reported that miRNA-21 positively regulates odontoblastic differentiation of hDP-SCs coordinating with STAT3 in the inflammatory microenvironment.
In our study, we collected inflammatory and healthy pulp tissues for differential expression profiles analysis to explore the genes that were overexpressed or silenced in the inflammatory conditions. We found that TAS2R was significantly up-regulated in the inflammatory pulp tissues and LPS-stimulated hDPSCs. TAS2R was found to be expressed in hDPSCs for the first time. TAS2R was initially identified in taste buds of the tongue and played a role in recognizing bitter taste, preventing the ingestion of toxic substances and performing self-defense functions [32]. There is increasing evidence that TAS2R has a variety of functions independent of taste in antiinflammation [16], anti-infection [14], reduction airway hyper-responsiveness [33]. Recent studies have reported that TAS2R plays an important role in the regulation of keratinocytes and cancer stem cells differentiation [11,12]. Therefore, it was reasonable that TAS2R increased to regulate the process of odontoblastic differentiation in hDPSCs. Grassin-Delyle et al. [16] reported that TAS2R expression is increased in lung macrophages from patients with pneumonia and plays an anti-inflammatory role by inhibiting the production of inflammatory cytokines (TNF-α, CCL3, CXCL8 and IL-10). The activation of TAS2R significantly inhibits the LPS-induced proinflammatory mediators release in human blood (TNFα, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17) [34]. In human gingival fibroblasts, LPS-induced pro-inflammatory cytokines (IL-6 and IL-8) are suppressed by TAS2R activation [13]. All of these studies suggest that TAS2R has a potential role in controlling inflammation. Pulpitis is characterized by the production of high levels of proinflammatory cytokines including TNFα, IL-4, IL-6, IL-8, IL-10 and CXCL8 [2,4]. We speculate that TAS2R promotion of pulp repair is not only related to the increase in hDPSCs differentiation, but also involved in its antiinflammatory function, implying its therapeutic potential for pulpitis in the early stage.
In the pulpitis, LPS, TNFα and IL-6 are released at high levels. Therefore, we stimulated hDPSCs to produce inflammatory microenvironment by LPS, TNFα or IL-6. We found that LPS, TNFα or IL-6 could increase the ALP, RUNX2, DMP-1 and DSPP expressions and promote odontoblastic differentiation of hDPSCs. Microarray results showed that ALP and RUNX2 were upregulated, suggesting the hDPSCs in the inflammatory pulp tissues had differentiation activity, because ALP and RUNX2 were markers of odontoblastic differentiation in hDPSCs at early stage and were highly expressed during the repair of dental pulp injuries [35,36]. But the clinically inflammatory pulp tissues used by microarray were the samples diagnosed as irreversible pulpitis. The initial odontoblasts in the inflammatory pulp have been partially damaged, and DMP-1 was secreted by the odontoblasts [37], so the expression of DMP-1 was down-regulated in the microarray results.
To better understand the role of TAS2R in odontoblastic differentiation of hDPSCs and pulp regeneration, TAS2R10 was overexpressed in hDPSCs, and the expression markers related to odontoblastic differentiation significantly increased. We also found that when TAS2R10 was silenced, the efficiency of odontoblastic differentiation of hDPSCs was reduced (compared with LPS + ODI group), but higher than that of ODI group. We speculate that inflammation promotes odontoblastic differentiation of hDPSCs may also involve other signal pathways like TLR4, ERK, P38 MAPK, AKT, or miRNA21/STAT3 [10,19], and inflammation activation of TAS2R10 is only one of these pathways. TAS2R and its downstream signaling have been found in multiple extraterritorial tissues throughout the body such as digestive system [38,39], reproductive system [40] and cardiovascular system [41]. Intracellular Ca 2+ elevation is a downstream effect mediated by TAS2R that plays crucial roles in numerous physiological and pathological activities. Deshpande et al. [33] have reported that TAS2R is expressed in the human airway smooth muscle cells and the activation of TAS2R increases intracellular Ca 2+ concentration, evokes airway relaxation and reduces airway hyper-responsiveness. TAS2R negatively regulates the release of thyroid-stimulating hormone through increasing Ca 2+ in thyrocytes [42]. Previous studies have also suggested that intracellular Ca 2+ positively regulates the odontoblastic differentiation of hDPSCs [27] and Ca 2+ levels regulate dentin formation in vitro and in vivo [28]. This is consistent with the results of our study that activation of TAS2R10 promoted odontoblastic differentiation of hDPSCs, which was indeed regulated by Ca 2+ .

Conclusions
In summary, our study demonstrated that TAS2R was found to be expressed in hDPSCs, and TAS2R regulated odontoblastic differentiation of hDPSCs by mediating intracellular Ca 2+ in inflammatory microenvironment, and shed a new light on hDPSCs-based therapeutic strategies for regenerative endodontics.